Fascin is a 55 kDa actin-bundling protein originally isolated from sea urchin egg actin gels. Our early studies demonstrated that fascin organized actin filaments into the bundles of sea urchin egg microvilli and coelomocyte filopodia. Our recent cloning of a cDNA for echinoid fascin led to cloning of this molecule from mouse, humans and Xenopus, and uncovered a Drosophila homologue in the singed gene. Early work on sea urchin fascin suggests a way to explain the Drosophila singed phenotype which includes deformed sensory bristles and female sterility. What previously was considered an 'exotic' invertebrate egg protein is turning out to be the most general and abundant actin-bundling protein in eukaryotic cells. None of the fascins are localized to 'classical microvilli' and they have no sequence similarity with villin, fimbrin, or with other known actin-binding proteins that are localized to these structures indicating the fascins are a novel family of actin-associated proteins. We have cloned, expressed and partially characterized an active mouse homologue of fascin. Westerns, Northerns, antibody localizations and in situ mRNA localizations indicate that fascin is most abundant in developing and adult neural tissue. In cultured neurons, fascin is found in filopodia and microspikes in growth cones where we believe it organizes the microfilament 'cores' of these protrusions which are proposed to act as sensors that sample the local environment for guidance cues that direct axonal growth. This proposal is centered on two main issues, one focused on neuronal cell biology; the other on fascin protein-protein interactions. I) Given the evidence that fascin is the actin-bundling protein found in nerve growth cone filopodia, we propose to determine if fascin is required for neurite outgrowth and/or neuronal guidance. Our preliminary experiments indicate fascin antisense oligonucleotides reduce fascin expression and affect neurite outgrowth in cultured neurons suggesting fascin is essential for outgrowth. 2) Growth cone filopodia are known to be highly dynamic structures remodeling on a timescale of minutes. F-actin-fascin interactions in vitro, on the other hand are quite stable. We propose to determine what regulates actin-fascin interactions. Specifically we are proposing to determine what protein(s) fascin interacts with in addition to F-actin. Our ongoing characterization of recombinant and native mouse fascin has shown there are several fascin isoforms which could be accounted for by phosphorylation or by modification of basic amino acids. Attempts to demonstrate phosphorylation of fascin have been unsuccessful suggesting acetylation or other modifications may be important. These isoforms do not appear to interact differentially with F-actin, however, suggesting other factors are involved in modulating this interaction.